ISSN 0975-6299
Vol.1/Issue-4/Oct-Dec.2010
International Journal of Pharma and Bio Sciences
ALTERATIONS IN THE PROFILE OF BLOOD CELLS OF WISTAR RATS INDUCED
BY LONG-TERM INGESTION OF CHLORPYRIFOS
SAROJNI TRIPATHI AND AJAI KUMAR SRIVASTAV*
Department of Zoology, DDU Gorakhpur University, Gorakhpur 273 009, India
*Corresponding Author
[email protected]
ABSTRACT
Male Wistar rats were divided into three groups -- A, B and C. Two groups (B and C) of animals were
daily administered orally chlorpyrifos at a dose of 5 and 10 mg/kg b wt., respectively. One group (A)
was employed as control. Rats were sacrificed 24 h after last dose on 1st, 2nd, 4th, 6th, and 8th week
after initiation of the experiment. Morphological alterations in blood cell profiles induced by
chlorpyrifos treatment were investigated on hematological preparations made by routine methods.
Results of the present study indicate that chlorpyrifos provoked an alteration of rat erythrocyte
morphology from the normal discoid shape to other forms such as echinocytes, dacrocytes,
schistocytes etc. Following chlorpyrifos treatment, few faded red blood cells were encountered. Many
cells were degenerated thus at places cell debris were present. Few nucleated red cells were also
seen.
KEY WORDS
Chlorpyrifos, Blood cells, Organophosphate, Rat
INTRODUCTION
Pesticides, a unique group of compounds, are
used to prevent, control or eliminate pests which
are a major cause of crop losses in the field as
well as in storage. These pests have always
been a nuisance and create multidimensional
problems for human beings. The discovery of
organic pesticides provided man with a new and
powerful weapon for his incessant war against
pests. Pesticides have benefited human beings
by controlling insects, disease transmitting
rodents, noxious arthropods and pests of plants
including crops and forests. But pesticides may
have dual actions – they are important in
controlling injurious pests but they may also
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present a hazard to species not considered to
be pests in the environment. This ill-effect
becomes
particularly
significant
when
pesticides directly affect populations of
economically important organisms or poison
organisms which are eaten by economically
important animals and human beings.
The effects of toxicants may be lethal or
sublethal. Lethal effects may result in death of
the organism (mostly in acute effects). A typical
lethal effect is failure of the chemically exposed
organisms to produce viable offspring’s. The
most common sublethal effects are behavioural
changes (e.g. swimming, attraction avoidance
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and prey-predator relationships), physiological
changes (e.g. growth, reproduction and
development), biochemical changes (e.g. blood
enzyme and ion levels) and histological changes.
Together,
behavioural,
biochemical,
physiological and histopathological tests are
useful for evaluating the environmental hazard of
a chemical, and they may provide important
information on its mode of action (Rand and
Petrocelli 1985).
The organophosphate insecticides have
superseded the organochlorines owing to their
rapid biodegradability and shorter persistence in
the environment. But as a consequence of their
indiscriminate and widespread use in agriculture
and public health, these insecticides ultimately
reach the environment and affect the life therein.
Organophosphate
compounds
have
neuroparalytic and enzymatic actions. The
toxicity of these compounds lies in the capacity
of their selective effect on enzymes of nerve
tissue – cholinesterase (ChE), which lead to
excessive accumulation of acetylcholine in the
organisms (Pan and Dutta1998; Quistad and
Casida 2000).
Chlorpyrifos
[O,O-diethyl-O-(3,5,6trichloro -2- pyridil) phosphoro-thioate], is a
member
of
organophosphate
class
of
insecticides that displays broad-spectrum
insecticidal activity against a number of
important arthropod pests (Racke 1993). It is
recommended as a most effective insecticide for
the control of mustard aphid, Liapaphis erysimi
(Srivastava and Srivastava 1986). The widespread use of chlorpyrifos is probably due to its
relatively low mammalian toxicity. Chlorpyrifos
has been reported to induce immune alterations
associated with lymphocyte subpopulations in
rats (Blakley et al. 1999).
Wang et al. (2009) have stated
chlorpyrifos is generally toxic to mammals,
however, the toxicity of this pesticide to other
organs and their potential interactive effects still
remain unclear. Blood is a pathological indicator
of the whole body, and hence hematological
parameters are important for analysis of the
functional status of an exposed animal to
suspected toxicant (Omitoyin 2006). Moreover,
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blood cell profile has been considered as an
important indicator of diseases and other
toxicants. As pathomorphological changes are
indicative of numerous diseases (Yawata
2003) there is little doubt of the importance of
elucidating
the
mechanism
governing
erythrocyte shape (Pawlowski et al. 2006). To
our knowledge there exists no report regarding
the effect of chlorpyrifos exposure to
mammals. Keeping above facts in view, the
present study was designed to evaluate the
effect of orally administered chlorpyrifos at
different doses for two months on the blood cell
profile of rat. Oral administration seems to be
the most appropriate in long-term exposure of
chlorpyrifos as it enters the animal or human
body through the residues in food. Also, oral
route for exposing chlorpyrifos in rats rather
than injecting the chemical would better reflect
the dietary exposure that most human
population experiences. Hence, the effects due
to repeated dose administration by gavage
have been investigated in this study.
MATERIALS AND METHODS
Wistar rats (males; b wt 145-165 g) were
procured from the Indian Institute of Toxicology
Research, Lucknow, India. The animals were
acclimatized for at least 2 weeks prior to
chlorpyrifos treatments. The rats were
maintained on the standard laboratory feed
and water ad libitum throughout the
experimental period.
After acclimation rats were separated
into three groups of 25 each. Animals in group
A (GA) served as control. Rats from group B
(GB) were daily administered orally chlorpyrifos
(Anu Products Ltd., India) at a dose of 5 mg/kg
b wt. Animals in group C (GC) received daily
an oral administration of chlorpyrifos at a dose
of 10 mg/kg b wt. Fresh dosing solutions of
chlorpyrifos
were prepared each day.
Chlorpyrifos treatments were given at 08:00
each day throughout the experiment as it has
been reported that efficacy of chlorpyrifos is
altered depending on whether it is
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administered in the morning or afternoon
(Kuperberg et al., 2000).
Five rats (from all the three groups) were
sacrificed 24 h after last dose on 1st, 2nd, 4th, 6th,
and 8th week after initiation of the experiment
under light ether anesthesia. The animals were
fasted overnight before sacrifice. Fresh blood
was collected (from each rat from all three
groups) on each interval from jugular vein. Blood
smears were prepared, dried in air and after
fixing for 10 minutes in methyl alcohol stained
with giemsa for 20 minutes. Stained preparations
were examined with microscope (Olympus CH
20i) for morphological changes in blood cells.
Photomicrographs were taken with the aid of
Olympus E 420 camera.
Observations
Group A:In control rats the erythrocytes (red
cells) were noticed as round, non-nucleated
biconcave discs having a central pallor which
covers about one third of the cell (Fig. 1).
Group B:Up to 2 weeks treatment with 5 mg/kg
b wt of chlorpyrifos there was no change in the
profile of the blood cells. After 4 week
chlorpyrifos exposure the red blood cells were
noticed to be increased in size (Fig. 2). Few red
blood cells with irregularly spaced projections
(acanthocytes) were noticed (Fig. 3). Teardrop
shaped red blood cells (dacrocytes) were also
found (Fig. 3). After 6 week chlorpyrifos
treatment, red blood cells almost spherical in
shape, were recorded possessing no area of
central pallor like a normal red blood cell
(spherocytes) (Fig. 4). Few fragmented red blood
cells (schistocytes) were also noticed (Fig. 5).
Following 8 week chlorpyrifos treatment, few
faded red blood cells were encountered (Fig. 6).
Many cells were degenerated thus at places cell
debris were present (Fig. 7).
Group C:There was no change in the profile of
the blood cells after 1 week treatment with 10
mg/kg b wt of chlorpyrifos. After 2 week
exposure few red blood cells increased in size.
Also, few red blood cells with irregularly spaced
projections (acanthocytes) and echinocytes were
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noticed (Fig. 8). Dacrocytes also mark their
presence (Fig. 9). Following 4 week treatment,
spherocytes,
schistocytes
and
many
echinocytes were noticed (Fig. 10). Few
degenerating red blood cells were also
observed (Fig. 11). Number of lymphocytes
and neutrophils increased. In 6 week
chlorpyrifos treated rats red blood cells
showing blue pigments (polychromatophilic)
were recorded (Fig. 12). Several spherocytes
(Fig. 13) were present in addition to
schistocytes, dacrocytes and acanthocytes
(Fig. 14). Many degenerating blood cells (red
blood cells and white blood cells) were also
recorded (Fig. 15). After 8 week chlorpyrifos
exposure there were present several
degenerating cells which clustered to form
debris (Fig. 16). Also several red blood cells
with an oval or rectangular area of central
pallor (stomatocytes) and with a central color
spot in the area of pallor (target cells) (Fig.17)
were noticed. Few nucleated red cells were
also seen (Fig. 18).
DISCUSSION
Results of the present study indicate that
chlorpyrifos provoked an alteration of
rat
erythrocyte morphology from the normal
discoid shape to other forms such as
echinocytes, dacrocytes,
schistocytes etc.
Echinocytes
transformation
plays
an
antihemolytic role – the expansion of the
plasma membrane increases the cell-volume
ratio/membrane-area, thus allowing swelling of
the cells before lysing (Isomaa et al. 1986).
Zeni et al. (2002) noticed occurrence of
echinocytes in Ictalurus melas following
exposure to an anionic detergent (sodium
dodecyl benzene sulphonate) and suggested
that echinocytes may result from cellular
adaptation
of
physiological
parameters
involved in shape maintenance. Suwalsky et al.
(2008) have reported that human erythrocytes
when incubated with aqueous extract of
Aristotelia chilensis showed an alteration in
erythrocyte morphology from the normal
discoid shape to an echinocytic form. They
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have postulated that cell membrane lipid bilayers
are in general potential targets for Aristotelia
chilensis polyphenols. The presence of
echinocytes (crenated cells) might influence the
blood flow by increasing viscosity, resulting from
the intermeshing of the spicules (Reinhart and
Chien
1986)
thus
obstructing
the
microcirculation.
In the present study fragmented red blood
cells
(schistocytes)
were
noticed
after
chlorpyrifos treatment to rats. This derives
support from the previous findings of several
investigators who have reported the occurrence
of schistocytes after treatment with aluminium
(rats – Vittori et al. 1999) and phenylhydrazine
(calf – Sharma et al. 1991). Structural defects
and changes in surface shapes of erythrocytes
have been reported by Koc et al. (2008) from
endosulfan and malathion exposed rats.
Changes in the erythrocyte profile were also
noticed in fishes by Benarji and Rajendranath
(1990 – dichlorvos), Tavares et al. (1999 –
trichlorphon), Khattak and Hafeez (1996 –
malathion), Singh and Srivastava (1994 –
formothion) and Sampath et al. (1993 – Ekolux
organophosphorus preparation). Cox et al.
(http://www.vet.uga.edu/vpp/clerk/cox/index.php)
have stated that schistocytes are one of the
poikilocytes used to monitor toxicity. Also they
have suggested glomerulonephritis as one of the
factors for the formation of schistocytes. We also
supports that kidney and liver lesions (our
unpublished results) observed after chlorpyrifos
treatment to rats may be the reason for the
presence of schistocytes. This can also be
considered as supportive data for the other cell
deformities (dacrocytes, acanthocytes) noticed in
the present study. Also, it can be speculated that
chlorpyrifos
caused
alterations
in
the
cytoskeleton (membrane proteins and/or lipids)
of red blood cells thus affecting the surface area
of the cell. Comelekoglu et al. (2000) stated that
some pesticides may provoke the alterations in
size and surface shapes of erythrocytes.
Nikimma (1992) suggested that toxic materials
directly or indirectly damage the membrane
structure, ion permeability and cell metabolism of
erythrocytes thus may cause morphologically
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damaged erythrocyte formation. Akahane et al.
(1987) have reported an increased cholesterol
and phospholipids in red cell membrane after
malotilate (a hepatotropic agent) treatment to
rats and suggested that malotilate causes an
increase in the surface area of the erythrocytes
by accelerating the incorporation of cholesterol
into their membrane and such erythrocyte
might be rheologically impaired and captured
more easily by the spleen.
In
chlorpyrifos
exposed
rats
polychromatophilic cells have been observed.
These cells have been considered to be
immature red blood cells and represent
accelerated red cell production due to stressed
bone marrow. Occurrence of nucleated red
blood cells in the present study indicates an
active marrow response releasing premature
release of normoblasts into circulation.
An increase in the cellular size of the
red blood cells has been recorded in rats postexposure to chlorpyrifos. This could be due to
the membrane alterations. Chlorpyrifos may
have impact on the cell flexibility and
permeability by mechanisms not yet defined.
The increased cellular size observed in this
study may derive support from the suggestions
of Vives et al. (1999) who explained that the
expansion of membrane increases the
area/volume proportion and could allow the
swelling of the cell, thus, reaching the largest
volume before the lysis. Swelling of red blood
cells as reflected by the increased mean
corpuscular volume (MCV) has been attributed
to the increase in the activity (Soivio et al.
1974). An increase of erythrocyte size (MCV)
has been associated with several factors but it
is generally considered as a response to stress
(Weber 1982).
In the present study, stomatocytes and
target cells have been observed in chlorpyrifos
exposed rats. Similar cells have also been
reported from rats treated with aluminium
(Vittori et al. 1999; Bazzoni et al. 2005) and
malotilate (Akahane et al. 1987). Vittori et al.
(1999) stated that presence of atypical cells
(target and crenated cells) induced by
aluminium ingestion to rats strongly suggests
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membrane alteration. Bazzoni et al. (2005) have
attributed the presence of stomatocytes in
aluminium treated rats, to an expansion of the
inner monolayer area, or to a shrinkage of the
external one. They further stated that the cavities
which give place to the stomatocytic shapes
indicate, generally, a stress of the membrane.
Chlorpyrifos treated rats showed the
degeneration of white blood cells. Svoboda et al.
(2001) observed decrease of non-specific
immunity after diazinon (organophosphorus)
exposure to fish Cyprinus carpio and attributed
this to decreased leucocyte count. Decreased
white blood cell count has also been reported
from teleosts exposed to diazinon (Koprucu et al.
2006; Adedeji et al. 2009), methylparathion
(Nath and Banerjee 1996) and cadmium (KoriSiakpere et al. 2006). Reduced number of white
blood cells has been noticed in chlorpyrifos
exposed bird (Obaineh and Matthew 2009).
These reports regarding the decreased white
blood cell count provide supportive evidence to
the present study in which degenerating white
blood cells have been noticed after toxicant
treatment to rats.
In this study, cellular debris formed by
degeneration of red blood cells has been
noticed in rats following post-exposure to
chlorpyrifos. Several studies in past have
reported decreased erythrocyte count and
hemoglobin content after toxicant exposure to
fish (Adhikari et al. 2004; Koprucu et al. 2006;
Kori-Siakpere et al. 2006; Patnaik and Patra
2006) and rat (Koc et al. 2008). In these
reports the decreased red blood cell
number/hemoglobin content may be accounted
for by the destruction of red blood
cells/hemolysis.
Under the light of this study, it is
concluded that chlorpyrifos is extremely
effective in causing alterations in red blood
cells and white blood cells. These changes
may be potentially harmful for the survival of
the organism if exposed for long-term to the
toxicant. Even though limitations exist to
extrapolate experimental results in animals to
humans, this study might be useful in carrying
in vitro investigations with human erythrocytes
to fully understand the possible deleterious
effects of the pesticide on structural alterations
and survival of these cells.
Fig. 1 Normal red blood cells with central pallor of control rat. x 500.
Fig. 2 Red blood cells of chlorpyrifos (5 mg/kg b wt) treated rat. Note increased size of the cells (arrows). x 500.
Fig. 3 Presence of acanthocytes (broken arrow) and dacrocytes (arrow) in the blood of chlorpyrifos (5 mg/kg b
wt) treated rat. x 500.
Fig. 4 Spherocytes (arrows) in the blood of rat treated with chlorpyrifos (5 mg/kg b wt). Note the absence of
central pallor in these cells. x 500.
Fig. 5 Fragmented red blood cell (schistocyte) in the blood of chlorpyrifos (5 mg/kg b wt) treated rat. x 500.
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Fig. 6 Faded red blood cells (arrows) in the rat exposed to chlorpyrifos (5 mg/kg b wt). x 500.
Fig. 7 Cell debris (arrow) formed by degeneration of cells in the chlorpyrifos (5 mg/kg b wt) treated rat. Mark
also the presence of dacrocyte (broken arrow) and schistocyte (arrow head). x 500.
Fig. 8 Acanthocyte (arrow) and echinocyte (broken arrow) in the blood of chlorpyrifos (10 mg/kg b wt) treated
rat. x 500.
Fig. 9 Schistocyte (broken arrow) and dacrocyte (arrow) in the rat exposed to chlorpyrifos (10 mg/kg b wt). x
500.
Fig. 10
Fig. 11
Fig. 12
Fig. 13
Fig. 14
Fig. 15
Fig. 16
Fig. 17
Fig. 18
Echinocytes (arrows) in the blood of chlorpyrifos (10 mg/kg b wt) treated rat. x 500.
Degenerating red blood cells (arrows) in the blood of chlorpyrifos (10 mg/kg b wt) treated rat. x 500.
Polychromatophilic cell (arrow) in chlorpyrifos (10 mg/kg b wt) exposed rat. x 500.
Spherocytes (arrows) in the blood of rat treated with chlorpyrifos (10 mg/kg b wt). Note the absence of
central pallor in these cells. x 500.
Acanthocytes (arrows) in the blood of chlorpyrifos (10 mg/kg b wt) treated rat. x 500.
Degenerating white blood cells (arrow) in the blood of chlorpyrifos (10 mg/kg b wt) treated rat.
x
500.
Cell debris (arrow) formed by degeneration of cells in the chlorpyrifos (10 mg/kg b wt) treated rat. x
500.
Target cells (arrows) and schistocytes (broken arrows) in the blood of chlorpyrifos (10 mg/kg b wt)
treated rat. x 500.
Nucleated red blood cell (arrow) in chlorpyrifos (10 mg/kg b wt) treated rat. x 500.
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